With an estimated one third of the world's population infected with Mycobacterium tuberculosis (Mtb) (i.e. more than two billion individuals) and 9 to 10 million new cases and 2 million deaths every year, tuberculosis (TB) is a global and worldwide health problem. Generally, person-to-person transmission occurs by aerosolized droplets generated by a person suffering from pulmonary TB (active disease). Among those infected (an estimated 30% of exposed individuals), only 5-10% will develop active TB disease within 2 years post-exposure (known as primary TB). However, the majority of infected individuals develop latent infection (LTBI) which can last decades without clinical signs or symptoms of disease. LTBI represents a state of equilibrium in which the infected subject is able to control the infection but not completely eradicate the bacteria. Reactivation (active TB after remote infection) may occur at a later stage, particularly in the elderly or in immunocompromised individuals as in the case of HIV infection and treatment with TNF inhibitors. The risk of TB reactivation is estimated as 10% per lifetime and impaired immunity increases the risk to 10% per year.
Mycobacterium tuberculosis (Mtb) bacillus, the causative agent of TB, possesses a circular genome of 4 411 529 base pairs (bp) which was fully sequenced in 1998 (Cole et al., 1998, Nature 393: 537-44). Mtb encodes approximately 4000 genes; however the function and role in Mtb life cycle and pathogenesis of the majority of these genes have not yet been elucidated.
Analysis of the genome sequences from closely related mycobacteria and comparative studies have permitted to identify a number of secreted proteins, including members of the Esx and PE/PPE gene families.
Although no structure or precise function is known for the various members of the PE/PPE families, it has been suggested that some of them may play a role in immune evasion, virulence and host specificity of the infecting Mycobacterium. Genome analyses revealed that the PE and PPE genes are frequently found adjacent in the Mtb genome and functionally linked (Riley et al., 2008, PLoS Comput Biol, 4:e1000174). It is thus assumed that such pairs of PE/PPE proteins (e.g. Rv2431c/Rv2430, Rv3477/Rv3478, etc) are interacting each other to form heterodimers which are likely the functional forms of these proteins.
As the PE and PPE gene family, the majority of Esx genes are expressed as tandem pairs that are coordinately regulated. The M. tuberculosis genome contains 23 EsX genes (named Esx A to W), which encode proteins presumably linked to Mtb virulence. Biophysical studies indicate that gene products of Esx pairs interact each other in functional heterodimers. For illustrative purpose, structural analysis of the TB9.8 (Rv0287)/TB10.4 (Rv0288) complex revealed that 19 amino acid residues from TB9.8 and 21 amino acid residues from TB10.4 are involved in the intramolecular contacts (Ilghari et al., 2011, J. Biol. Chem., 286: 29993-30002).
Previous attempts to overexpress Mtb EsxA (ESAT6) and EsxB (CFP 10) proteins of M. tuberculosis individually in E. coli were hampered by technical difficulties which resulted in low yields of protein. Several studies tend to indicate that expression of related protein pairs together would facilitate appropriate folding and dimerization permitting high yields of recombinant protein to be produced which simplify structural and biochemical studies of these protein families involved in Mtb virulence (Strong et al., 2006, Proc. Natl. Acad. Sci, 103: 8060-5; Mehra et al., 2013, PLoS, 9: e1003734). However, there is no indication that such dimers retain immunogenic activities.
Mtb-caused million deaths every year are particularly dramatic considering that both vaccine (Bacille-Calmette-Guérin (BCG)) and antibiotics exist and are widely used. However, if BCG appears to be effective at preventing disease in newborns and toddlers, it does not protect adults and fails to prevent Mtb reactivation in latently infected persons. On the other hand, treatment of active TB with various antibiotic combinations appears efficacious but requires strong patient compliance with daily administrations of different drugs over several months. Moreover, there is an alarming rate of appearance of drug resistant Mtb strains (e.g. “MultiDrug Resistant” (MDR), “eXtensively Drug-Resistant” (XDR) and “Totally Drug Resistant” (TDR) strains), mostly because of improper observance of this lengthy and costly drug regimen treatment.
There are several lines of evidence suggesting that stimulation of the cellular immune system plays a role in controlling TB disease (Rook et al., 2007, J Infect Dis, 196:191-8). The central role of CD4 T lymphocytes to control the pathogen and to prevent progression to disease is well established. For instance, HIV/AIDS patients with low CD4+ T cells count are more susceptible to progression to TB disease while antiviral treatments that elevate CD4+ T cells reduce progression to TB disease. However, CD4 T cells do not operate alone and are supported by CD8 T cells and other T cell subsets.
Development of effective TB vaccines is therefore a priority in this worrying context and two main approaches are being investigated for the last decade: replacement of BCG and BCG booster.
BCG replacement candidates aim at improving BCG efficacy and safety and are mainly based on live attenuated bacteria such as genetically modified BCG or Mtb strains engineered to express new sets of antigens that are absent from BCG or to overexpress Mtb antigens that BCG expresses but at a likely insufficient level or still to delete virulence genes and their regulators (e.g WO2009/064825; WO2012031752).
BCG boosters aim at inducing cellular and/or humoral immune responses and generally rely on recombinant vaccines designed for providing various TB antigens, either as protein composition generally admixed with potent Th1-activating adjuvants or through viral expressing vectors (see Andersen, 2007, Nature, 5: 484; Ottenhoff and Kaufman, 2012, PLoS 8(5): e1002607; Cayabyab et al., 2012, Frontiers in Cellular and Infection Microbiology 2: 1-16; and Brennan et al., 2012, Int J Tuberc. Lung Dis. 16(12): 1566-1573).
Some of these vaccine candidates have produced results in preclinical and clinical studies that demonstrate an ability to induce a robust cellular mediated immune response against Mtb or to provide protection against TB-associated lung lesions. For example, an adenoviral vector expressing Ag85A, TB10.4, TB9.8 and Acr2 (AdTBF) improved the effects of BCG, reducing lesion volume and bacterial load in the lungs of vaccinated goats (Perez de Val et al., 2013, PLoS, 8: e81317). However, these studies have highlighted the influence of various factors on the T cell response and protective efficacy such as the antigen doses (e.g. Aagaard et al., 2009, PLoS One, 4: 1-8) and administration routes (Goonetilleke et al., 2003, J. Immunol., 171: 1602-9).
The use of fusion polypeptides comprising various TB antigens has also been described. For example, the fusion protein Hyvac 4 (H4), which consists of Ag85B fused to TB10.4 (Aagaard et al., 2009, PLoS One, 4: 1-8) is in clinical development. The GSK's M72 fusion protein made of Rv1196 inserted in the middle of the serine protease Rv0125 showed a favorable clinical profile in terms of safety and immunogenicity when administered with different synthetic adjuvants (Von Eschen et al., 2009, Hum Vaccine, 5: 475-82). One may also cite the so-called “ID” fusion proteins (WO2008/124647) such as ID83 made of Rv1813, Rv3620 and Rv2608 and ID93 including Rv3619 fused to the three ID83 antigens as well as fusions of Rv0198 antigen with either Rv3812 or Rv0111 (see WO2011/144951). On the other hand, WO2014/009438 describes large fusions involving numerous mycobacterial antigens representative of all phases of the natural course of infection.
Despite all these and other efforts, tuberculosis is far from being controlled and there remains a need for alternative vaccine candidates for diagnosing, preventing and treating tuberculosis, especially in endemic regions.
The present invention fulfils this and other needs by providing an immunogenic combination which comprises at least heterooligomeric mycobacterial antigens preferably in fusion, which are selected from the group of the Esx, PE and PPE antigens. The combination/fusion of pairs of mycobacterial antigens involved in such heterooligomers (e.g. heterodimers) offers unexpected properties such as improvement of the antigen folding and the solubility of the fused antigens as compared to the individual antigens, which may increase genetic stability of the vaccine candidate, decrease potential cytotoxicity when produced in host cell or organism and/or improve quality and/or scope of the anti-Mycobacterium immunogenic response, whether humoral and/or cellular. In addition, the immunogenic combination of the invention may be tailored for different phases of the natural course of Mycobacterium infection with additional mycobacterial antigens. The present invention is particularly useful in the context of immunotherapy as stand-alone or as BCG booster for preventive or therapeutic purposes in the Mycobacterium infection field, e.g. preventing Mtb infection and/or prevention of primary TB and/or prevention of reactivation in latently infected subjects. It can also be used in association with standard (e.g. antibiotic-therapy) or any other novel treatment that is currently developed (e.g. small direct or indirect inhibitor molecules; antibodies or immunotherapeutics, etc). The present invention would also be helpful in the veterinary field, for example to reduce or abolish the risk of Mycobacterium infection and/or active disease in animals, especially in bovine and goat breedings.
This technical problem is solved by the provision of the embodiments as defined in the claims.
Other and further aspects, features and advantages of the present invention will be apparent from the following description of the presently preferred embodiments of the invention. These embodiments are given for the purpose of disclosure.